Although discovered for varying biological activities, ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), oncostatin M (OSM) and interleukin-6 (IL-6) comprise a defined family of cytokines (referred to herein as the xe2x80x9cCNTF familyxe2x80x9d of cytokines). These cytokines are grouped together because of their distant structural similarities [Bazan, J. Neuron 7: 197-208 (1991); Rose and Bruce, Proc. Natl. Acad. Sci. USA 88: 8641-8645 (1991)], and, perhaps more importantly, because they share xe2x80x9cxcex2xe2x80x9d signal-transducing receptor components [Baumann, et al., J. Biol. Chem. 265:19853-19862 (1993); Davis, et al., Science 260: 1805-1808 (1993); Gearing et al., Science 255:1434-1437 (1992); Ip et al., Cell 69: 1121-1132 (1992); Stahl, et al., J. Biol. Chem. 268: 7628-7631 (1993); Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. Receptor activation by this family of cytokines results from either homo- or hetero-dimerization of these xcex2 components [Davis, et al. Science 260: 1805-1808 (1993), Murakami, et al., Science 260: 1808-1810 (1993); Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. IL-6 receptor activation requires homodimerization of gp130 [Murakami, et al. Science 260: 1808-1810 (1993), Hibi, et al., Cell 63: 1149-1157 (1990)], a protein initially identified as the IL-6 signal transducer [Hibi, et al., Cell 63: 1149-1157 (1990)]. CNTF, LIF and OSM receptor activation results from heterodimerization between gp130 and a second gp130-related protein known as LIFRxcex2 [Davis, et al., Science 260: 1805-1808 (1993)], that was initially identified by its ability to bind LIF [Gearing et al., EMBO J. 10: 2839-2848 (1991)].
In addition to the xcex2 components, some of these cytokines also require specificity-determining xe2x80x9cxcex1xe2x80x9d components that are more limited in their tissue distribution than the xcex2 components, and thus determine the cellular targets of the particular cytokines [Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. Thus, LIF and OSM are broadly acting factors that may only require the presence of gp130 and LIFRxcex2 on responding cells, while CNTF requires CNTFRxcex1 [Stahl and Yancopoulos, Cell 74: 587-590 (1993)] and IL-6 requires IL-6Rxcex1 [Kishimoto, et al., Science 258: 593-597 (1992)]. Both CNTFRxcex1 (Davis et al., Science 259:1736-1739 (1993) and IL-6Rxcex1 [Hibi, et al. Cell 63:1149-1157, Murakami, et al., Science 260:1808-1810 (1990); Taga, et al., Cell 58:573-581 (1989)] can function as soluble proteins, consistent with the notion that they do not interact with intracellular signaling molecules but that they serve to help their ligands interact with the appropriate signal transducing xcex2 subunits [Stahl and Yancopoulos, Cell 74: 587-590 (1993)].
Additional evidence from other cytokine systems also supports the notion that dimerization provides a common mechanism by which all cytokine receptors initiate signal transduction. Growth hormone (GH) serves as perhaps the best example in this regard. Crystallographic studies have revealed that each GH molecule contains two distinct receptor binding sites, both of which are recognized by the same binding domain in the receptor, allowing a single molecule of GH to engage two receptor molecules [de Vos, et al., Science 255: 306-312 (1992)]. Dimerization occurs sequentially, with site 1 on the GH first binding to one receptor molecule, followed by the binding of site 2 to a second receptor molecule [Fuh, et al., Science 256: 1677-1680 (1992)]. Studies with the erythropoietin (EPO) receptor are also consistent with the importance of dimerization in receptor activation, as EPO receptors can be constitutively activated by a single amino acid change that introduces a cysteine residue and results in disulfide-linked homodimers [Watowich, et al., Proc. Natl. Acad. Sci. USA 89:2140-2144 (1992)].
In addition to homo- or hetero-dimerization of xcex2 subunits as the critical step for receptor activation, a second important feature is that formation of the final receptor complex by the CNTF family of cytokines occurs through a mechanism whereby the ligand successively binds to receptor components in an ordered manner [Davis, et al. Science 260:1805-1818 (1993); Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. Thus CNTF first binds to CNTFRxcex1, forming a complex which then binds gp130 to form an intermediate (called here the xcex1xcex21 intermediate) that is not signaling competent because it has only a single xcex2 component, before finally recruiting LIFRxcex2 to form a heterodimer of xcex2 components which then initiates signal transduction. Although a similar intermediate containing IL-6 bound to IL-6Rxcex1 and a single molecule of gp130 has not been directly isolated, we have postulated that it does exist by analogy to its distant relative, CNTF, as well as the fact that the final active IL-6 receptor complex recruits two gp130 monomers. Altogether, these findings led to a proposal for the structure of a generic cytokine receptor complex (FIG. 1) in which each cytokine can have up to 3 receptor binding sites: a site that binds to an optional xcex1 specificity-determining component (xcex1 site), a site that binds to the first xcex2 signal-transducing component (xcex21 site), and a site that binds to the second xcex2 signal-transducing component (xcex22 site) [Stahl and Yancopoulos, Cell 74: 587-590 (1993)]. These 3 sites are used in sequential fashion, with the last step in complex formationxe2x80x94resulting in xcex2 component dimerizationxe2x80x94critical for initiating signal transduction [Davis, et al. Science 260:1805-1818 (1993)]. Knowledge of the details of receptor activation and the existence of the non-functional xcex21 intermediate for CNTF has led to the finding that CNTF is a high affinity antagonist for IL-6 under certain circumstances, and provides the strategic basis for designing ligand or receptor-based antagonists for the CNTF family of cytokines as detailed below.
Once cytokine binding induces receptor complex formation, the dimerization of xcex2 components activates intracellular tyrosine kinase activity that results in phosphorylation of a wide variety of substrates [Ip, et al. Cell 69:121-1132 (1992)]. This activation of tyrosine kinase appears to be critical for downstream events since inhibitors that block the tyrosine phosphorylations also prevent later events such as gene inductions [Ip, et al., Cell 69:121-1132 (1992); Nakajima and Wall, Mol. Cell. Biol. 11:1409-1418 (1991)]. Recently, we have demonstrated that a newly discovered family of non-receptor tyrosine kinases that includes Jak1, Jak2, and Tyk2 (referred to as the Jak/Tyk kinases) [Firmbach-Kraft, et al., Oncogene 5:1329-1336 (1990); Wilks, et al., Mol. Cell. Biol. 11: 2057-2065 (1991]and that are involved in signal transduction with other cytokines [Argetsinger, et al., Cell 74:237-244 (1993); Silvennoinen, et al., Proc. Natl. Acad. Sci. USA 90:8429-8433 (1993); Velazquez, et al., Cell 70: 313-322 (1992); Witthuhn, et al., Cell 74:227-236 (1993)], preassociate with the cytoplasmic domains of the xcex2 subunits gp130 and LIFRxcex2 in the absence of ligand, and become tyrosine phosphorylated and activated upon ligand addition [Stahl et al., Science 263:92-95 (1994)]. Therefore these kinases appear to be the most proximal step of intracellular signal transduction activated inside the cell as a result of ligand binding outside of the cell. Assay systems for screening collections of small molecules for specific agonist or antagonist activities based on this system are described below.
The CNTF family of cytokines play important roles in a wide variety of physiological processes that provide potential therapeutic applications for both antagonists and agonists.
An object of the present invention is the production of cytokine antagonists that are useful in the treatment of cytokine related diseases or disorders.
Another object of the invention is the use of the disclosed cytokine antagonists for the treatment of cytokine related diseases or disorders. For example, an IL-6 antagonist described herein may be used for the treatment of osteoporosis, the primary and second effects of cancers, including multiple myeloma, or cachexia.
Another object of the invention is the development of screening systems useful for identifying novel agonists and antagonists of cytokine receptors. Another object of the invention is the development of screening systems useful for identifying small molecules that act as agonists or antagonists of the cytokines.
Another object of the invention is the development of screening systems useful for identifying novel agonists and antagonists of members of the CNTF family of cytokines.
Another object of the invention is the development of screening systems useful for identifying small molecules that act as agonists or antagonists of the CNTF family of cytokines.